Over the past 10 years it has become increasingly apparent that there is exceptional complexity in the RNA population in cells, including microRNAs, Piwi-interacting RNAs, termini-associated RNAs and other noncoding RNAs. In many cases, alterations in these RNAs, or proteins that bind to these RNAs, have been linked to a wide range of medical disorders. A major challenge of molecular biology is to determine the function of these fascinating and novel RNA species. In order to understand how these RNAs function in cells, it will be exceptionally valuable to be able to image the localization and intracellular movements of these RNAs in living cells under a variety of experimental stimuli. We have developed a novel genetically encodable system to fluorescently tag RNAs in cells. This system utilizes an RNA sequence element, termed Spinach, which is appended to an RNA of interest, and which "switches on" the fluorescence of an otherwise non-florescent dye. This dye is based on the structure of the fluorophore in GFP, making Spinach an RNA mimic of GFP. In this proposal, we will develop novel contamerization and stabilization strategies to make Spinach simple to use and highly sensitive for imaging of individual RNAs. The results of this phase I SBIR application will be RNA sequences and proof- of-principle data which will set the stage for the development of a commercial, plasmid based system for expressing fluorescently tagged RNAs in cells. Based on the past history of successful commercialization of GFP expression systems, we expect that this expression system will have high commercial potential, and the ability to provide an enabling technology to the larger research community.
The human genome encodes a large number of mRNAs and noncoding RNAs. Although many RNAs are linked to disease processes, cellular mechanisms by which these RNAs function in cells are not fully clear. This project will develop a toolkit whic will enable researchers to easily image both mRNAs an noncoding RNAs in living cells using a novel genetically encoded fluorescent RNA tagging system, thereby providing insights into the function of these RNAs in cell biology and disease pathogenesis.!